Human tissue-type plasminogen activator (t-PA) is a key physiological regulator of fibrinolysis. It converts the zymogen plasminogen into plasmin, the enzyme which degrades the fibrin network of the thrombus. Apparently, in the presence of a clot, both t-PA and plasminogen bind to fibrin and form a ternary complex in which plasminogen is efficiently activated. The affinity for fibrin makes t-PA clot-specific, and useful as a therapeutic agent for fibrinolytic therapy in man.
The principal physiological regulator of t-PA appears to be a specific, fast-acting, plasminogen activator inhibitor type-1 (PAI-1). PAI-1 is a protein of Mr 50,000 which binds to t-PA in a 1:1 complex, and inactivates the serine protease. Recent clinical studies suggest that elevated levels of PAI-1, by reducing the net endogenous fibrinolytic capacity, may contribute to the pathogenesis of various thrombotic disorders, including myocardial infarction, deep vein thrombosis, and disseminated intravascular coagulation.
The existence of two forms of PAI-1 differing in t-PA inhibitory activity and referred to as active and inactive or latent (inactive/latent), has been described previously. PAI-1 isolated from many mammalian cell types is obtained in a partially active latent form; the latent form is known to be converted to a more active form, by treatment with denaturants such as sodium dodecylsulfate (SDS). In addition, both active and inactive/latent forms of PAI-1 expressed in E. coli have been reported, as discussed below.
The expression in bacteria and purification of recombinant human PAI-1 cDNA has previously been reported. Ny et al. (1986) Proc. Natl. Acad. Sci. USA 83:6776-6780 disclosed the expression of functional rPAI-1 in E. coli, using a phage lambda gt11-derived vector. rPAI-1 was expressed as a beta-galactosidase-PAI-1 fusion protein of about 180 kDa, with the PAI-1 coding sequence fused to the E. coli beta-galactosidase coding sequence. Wun and Kretzmer (1987) FEBS Letters 210:11-16 similarly used a lambda gt11 expression vector to express functional rPAI-1 in E. coli. Pannekoek et al. (1986) EMBO J. 5:2539-2544 reported the expression of a functional 43 kDa form of rPAI-1 in E. coli using a pUC9-derived vector, as assayed by reverse fibrin autography. Although Ny et al., Pannekoek et al., and Wun and Kretzmer report the expression of functional rPAI-1 in E. coli, they did not report the purification of the E. coli -expressed rPAI-1 and did not quantitate the specific activity of the crude preparations of rPAI-1.
It has been reported from the Pannekoek group that rPAI-1 is expressed in E. coli almost exclusively in an inactive or latent form (Lambers et al. Fibrinolysis (1988) 2, Supp. 1:33). Apparently the 43 kDa form reported by Pannekoek et al. (1986) EMBO J. 5:2539-2544 is predominantly inactive. Another recent report states that rPAI-1 expressed in E. coli has biological activity toward urokinase almost equal to that of human fibrosarcoma PAI-1 (Lawrence et al. Fibrinolysis (1988) 2, Supp.1:54).
We previously reported the production of substantially pure, biologically functional, nonfused mature form E. coli-expressed human recombinant PAI-1 (rPAI-1) protein having a specific activity of about 300,000 units/mg, where a unit is defined as the amount of protein required to neutralize 1 international unit of t-PA in an S2288 chromogenic assay, where the enzymatic activity of t-PA to generate plasma from its plasminogen precursor is measured (Reilly et al., (1990) J. Biol. Chem. 265:9570-9574; copending, commonly assigned U.S. Ser. No. 07/350264, filed May 11, 1989). The biological activity of this preparation of PAI-1, was not significantly enhanced by treatment with protein denaturants.
We have previously reported the purification of rPAI-1 using a process including Q-Sepharose (anion exchanger) and S-Sepharose (cation exchanger) chromatography steps (Reilly et al., J. Biol. Chem. (1990) 265:9570-9574; U.S. Ser. No. 07/350,264, filed May 11, 1989). However, it was not suggested in these references that such ion exchange chromatography steps could be used to provide the resolution and separation of active form and inactive/latent form rPAI-1.
The separation of active and inactive/latent forms of PAI-1 using a single chromatographic step has not previously been disclosed. Indeed, the inability of previous researchers to resolve the active and inactive/latent forms of PAI-1 has presented a major difficulty in obtaining homogeneous active form PAI-1, or PAI-1 compositions of defined proportion of active form and inactive/latent forms. For example, Franke et al. Biochim. Biophys. Acta (1990) 1037:16-23) report that "Although isolation of active PAI-1 was of interest in principle, the pronounced lability of the inhibitory activity was considered to make this an unrealistic goal". Franke et al. consequently only reported a partly purified preparation of active form rPAI-1.
Lawrence et al. (Eur. J. Biochem. (1989) 186:523-533) reported the production and purification of rPAI-1. The purified E. coli-expressed rPAI-1 of Lawrence et al. was reported to have a specific activity similar to activated eukaryotic-expressed (natural or recombinant) PAI-1 and a percentage of the maximum theoretical inhibition of about 50%. Thus, the preparation E. coli-expressed rPAI-1 of Lawrence et al. appears to contain a mixture of active and inactive/latent forms. Significantly, Lawrence et al. do not report the resolution of active and inactive/latent form rPAI-1 using a chromatographic process. Lawrence et al. use the following chromatography steps: gel filtration (Sephacryl S-200), hydroxylapatite, and heparin-agarose. Indeed, Lawrence et al. report that the inactive/latent form and the activated form (guanidine-treated) of eukaryotic rPAI-1 have identical elution profiles on gel filtration chromatography.